Noninvasive Remote-Controlled Release of Drug Molecules in Vitro UsingMagnetic Actuation of Mechanized Nanoparticles

Courtney R. Thomas,†,‡ Daniel P. Ferris,†,‡ Jae-Hyun Lee,§ Eunjoo Choi,§ Mi Hyeon Cho,§

Eun Sook Kim,| J. Fraser Stoddart,⊥ Jeon-Soo Shin,| Jinwoo Cheon,*,§ and Jeffrey I. Zink*,†,‡

Department of Chemistry and Biochemistry and California NanoSystems Institute, UniVersity of California,Los Angeles, California 90095-1569, Department of Chemistry, Yonsei UniVersity, 262 Seongsanno Seodaemun-Gu,120-752 Seoul, Korea, Department of Microbiology, College of Medicine, Yonsei UniVersity, 120-749 Seoul, Korea,

and Department of Chemistry, Northwestern UniVersity, 2145 Sheridan Road, EVanston, Illinois 60208-3113

Received March 30, 2010; E-mail: jcheon@yonsei.ac.kr; zink@chem.ucla.edu

Abstract: Mesoporous silica nanoparticles are useful nanoma-terials that have demonstrated the ability to contain and releasecargos with mediation by gatekeepers. Magnetic nanocrystalshave the ability to exhibit hyperthermic effects when placed inan oscillating magnetic field. In a system combining these twomaterials and a thermally sensitive gatekeeper, a unique drugdelivery system can be produced. A novel material that incorpo-rates zinc-doped iron oxide nanocrystals within a mesoporoussilica framework that has been surface-modified with pseudoro-taxanes is described. Upon application of an AC magnetic field,the nanocrystals generate local internal heating, causing themolecular machines to disassemble and allowing the cargos(drugs) to be released. When breast cancer cells (MDA-MB-231)were treated with doxorubicin-loaded particles and exposed toan AC field, cell death occurred. This material promises to be anoninvasive, externally controlled drug delivery system withcancer-killing properties.

Mesoporous silica nanoparticles (MSNs) have attracted wide-spread research interest as functional materials.1-7 They areendocytosed by cells,1 are nontoxic,2 and can be used to deliverdrugs.3 Recently, an amazing array of methods for controlling poresto trap and release cargos have been developed. These range fromcoatings on particles to intricate nanovalves that control the poreopenings using light,4 pH,5 or redox6 activation. For therapeuticapplications, an external and noninvasive method of actuation ispreferable for control of therapeutic effects. Light control has beendemonstrated, but its practical applicability is limited because ofshallow tissue penetration for photodynamic therapies. Nanovalvesbased on changes in pH are self-opening but cannot be controlledby an external stimulus. This lack of an effective, external controlfor in vivo applications can be overcome by a new class of materialsdriven by a magnetic core.

Magnetic nanocrystals (NCs) are of importance in biomedicalapplications, as they can be used for both therapeutics and imaging.The usefulness of magnetic materials for inducing hyperthermiceffects when placed in an oscillating magnetic field7 and for T2-weighted magnetic resonance imaging (T2MRI) contrast8 makemagnetic NCs theranostic. Among those developed, zinc-doped ironoxide nanocrystals (ZnNCs)9 improve upon existing materials by

offering a 4-fold increase in hyperthermic effects and a roughly10-fold increase in MRI contrast relative to undoped iron oxideNCs.

We have combined the advantages of mechanized silica (MSNswith nanovalves) with those of zinc-doped iron oxide (Figure 1) tocreate a new generation of drug delivery systems responsive to heatactivation. To this effect, a nanovalve was chosen that is non-self-opening in biological systems, exhibits thermal stability at roomtemperature, and can be operated under heating.

When this nanovalve is attached to the surface of a mesoporousparticle, an increase in temperature causes the valve to open,allowing materials contained within to diffuse out. If the nanopar-ticles contain ZnNCs, then application of an oscillating magneticfield induces local heating, which should result in the same drug-release effect. This novel approach to drug delivery allows cargoto be contained within the nanoparticle at body temperature butresults in controlled release of the therapeutic agent to induceapoptosis upon local heating generated by the ZnNCs.

In this study, we discuss four experiments performed on thismagnetically activated release system (MARS): (1) macroscopicheating of the solution to induce guest release; (2) magnetic heatingvia application of an oscillating magnetic field as an external control;(3) localized magnetic heating without increasing the solutiontemperature in a thermostatted medium; and (4) remote-controlledactuation of the nanovalves to demonstrate controlled drug deliveryin cancer cells.

Magnetic-core silica nanoparticles (MCSNs, Scheme 1) weresynthesized by modifying a standard MCM-41-type synthesis.2c Tocontain the ZnNCs within the silica cores, the ZnNCs were firststabilized in a surfactant solution. The silica precursor tetraethylorthosilicate (TEOS) was added to a solution containing thecetyltrimethylammonium bromide (CTAB)-stabilized ZnNCs withsodium hydroxide. The base catalyzed the hydrolysis of the silicaprecursor to form the mesostructured nanoparticles around theZnNCs. Particle characterization confirmed the size and porediameter, and inclusion of ZnNCs was confirmed by transmission

† Department of Chemistry and Biochemistry, University of California, LosAngeles.

‡ California NanoSystems Institute, University of California, Los Angeles.§ Department of Chemistry, Yonsei University.| Department of Microbiology, Yonsei University.⊥ Northwestern University.

Figure 1. Electron micrographs of (a) zinc-doped iron oxide nanocrystals(ZnNCs) and (b, c) ZnNCs encapsulated within mesoporous silica.

Published on Web 07/16/2010

10.1021/ja1022267 2010 American Chemical Society J. AM. CHEM. SOC. 2010, 132, 10623–10625 9 10623

electron microscopy (Figure 1 and Table S1 and Figure S1 in theSupporting Information). To assemble the nanovalve for facilitationof magnetic actuation, a molecular machine was assembled on theparticle. N-(6-N-Aminohexyl)aminomethyltriethoxysilane was firstcondensed on the particle surface. Cargo loading was accomplishedby soaking the nanoparticles in a saturated solution of rhodamineB or doxorubicin to fill the mesoporous structure by diffusion,resulting in a 4 wt % loading. Containment of cargo in the poreswas achieved by adding cucurbit[6]uril, which electrostatically bindsthe molecular thread on the silica nanoparticle surface to the interiorof the 1 nm cyclic cucurbit[6]uril cavity.5b,10 After this step, theMARS nanoparticles were washed thoroughly with water to removeexcess dye adsorbed on the silica surface.

A nanovalve was selected for the MARS that remained closedat physiological temperature and opened when heated. The valveswere attached to the surfaces of MSNs without magnetic cores,and external heat was applied (Figure S3). At room temperature,the valves remained closed, and as the applied temperature wasincreased, dye was released.

The complete MARS was tested to determine whether magneti-cally induced heating would open the nanovalves, causing therelease of the contained fluorescent molecules. To perform thisstudy, MARS particles at room temperature were placed into anoscillating magnetic field, and dye release was observed as afunction of time. Although the source of heat was changed froman external source to the internal heating caused by magneticactuation, dye release was still observed (Figure 2a).

A sample of MCSNs was placed into an oscillating magneticfield to measure their effect on solution temperature. A sample ata concentration of 10 mg/mL was placed inside a water-cooledcopper coil producing an alternating current (AC) magnetic fieldhaving a frequency of 500 kHz and a current amplitude of 37.4kAm-1 (Taeyang Instrument Company, Korea). The temperatureof the 1 mL of water above the particles was monitored and foundto increase to a maximum temperature of 52 °C (Figure S2). Thiseffect was also observed for the ZnNCs in solution.9

For therapeutic applications, it is important to know whether theopening of the nanovalve is a result of internal heating of thenanoparticle or an increase in the ambient temperature. To determine

whether internal heating alone causes the valve to open, a sampleof the MARS was kept at 0 °C and placed into the oscillatingmagnetic field. The MARS was then activated by applying 1 minpulses of the AC field, and dye release was monitored using smallaliquots of the particle solution placed in a fluorometer. A singlepulse caused 40% of the rhodamine B dye to be released with adramatic increase in solution fluorescence (b in Figure 2b), whichwe attribute to rapid internal particle heating and valve opening. Asecond sample, pulsed intermittently, not only showed the sameinitial release of cargo but also continued dye release upon eachadditional pulse (9 in Figure 2b). It is clear that under theseconditions, the local internal heating is important for dye releasebut macroscopic heating of the bulk solution is not necessary forvalve actuation.

These materials are useful for in vitro drug delivery, asdemonstrated by the release of anticancer drugs in the breast cancercell line MDA-MB-231 (Figure 3a). The MARS nanoparticles weretaken up by the cells, and minimal drug release was observedbecause the surface-attached valves were closed (Figure 3a, images1 and 2). In the presence of the oscillating field, the local heatingcaused by the magnetic ZnNCs facilitated the release of doxorubicinfrom the silica pores, inducing apoptosis in the breast cancer cells(Figure 3a, images 5 and 6). In the images taken after a 5 minexposure to the magnetic field, a dramatic increase in intensity from

Scheme 1. Sketch of Nanoparticles, Machines, and Assemblya

a ZnNCs (1) are synthetically positioned at the core of the mesoporoussilica nanoparticles (2). The base of the molecular machine is then attachedto the nanoparticle surface (3). Drug is loaded into the particle and capped(4) to complete the system. Release can be realized using remote heatingvia the introduction of an oscillating magnetic field (5). The particles andmachines are not drawn to scale.

Figure 2. Cargo release using magnetic actuation. In (a), the MARSnanoparticles were continuously exposed to the magnetic field. The insetshows the data as a release profile. In (b), a sample was kept at 0 °C andexposed to pulses of the magnetic field. A single AC magnetic field exposure(b) exhibited ∼40% cargo release after an initial 1 min pulse. Multiplepulses performed at 1, 3, 5, 7, and 9 min and then every 20 min for 270min (9) enabled more dye release until all of the dye diffused out. A baseline(2) was obtained by monitoring the fluorescence with no pulses. The lowtemperature of the surrounding solution (0 °C) was maintained in order toobserve the effects only from the magnetic field and not from heating ofthe surrounding solution.

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the doxorubicin (red color) was seen as a result of drug deliveryinto the cells.

The effect of the MARS on the cells was examined without drugloading under the same conditions as those with drug-loadedparticles. When a sample not containing doxorubicin was endocy-tosed into the cells and exposed to the oscillating magnetic field,16% cell killing was observed, while 37% cell killing resulted fromexposure to the magnetic field when doxorubicin was contained inthe mesopores (Figure 3b). Thus, both hyperthermia and drugdelivery contributed to cell death.

In summary, we have demonstrated that novel magnetic-coresilica nanoparticles are effective in actuating nanovalves andreleasing anticancer drugs upon exposure to an oscillating magneticfield. Additionally, we have shown the feasibility of using thissystem as a drug delivery system in cancer cells. Optimization tobalance the hyperthermic and apoptotic effects by varying the lengthof the magnetic actuation is under investigation.

Acknowledgment. The authors thank Monty Liong and SarahAngelos for discussions and help in testing the theoretical andexperimental framework for this study. This work was supportedin part by the UC Toxic Substances Training and Research Program,the National Science Foundation (NSF CHE 0809384), the Nano-medical National Core Research Center (R15-2004-024-00000-0),the Creative Research Initiative (2010-0018286), the National

Research Laboratory (M10600000255), the World Class UniversityProgram, a KOSEF grant funded by the Korean Government (2009-00810010), and Second Stage BK21 Project for Chemistry andMedical Sciences of Yonsei University.

Supporting Information Available: Experimental and spectralcharacterization data. This material is available free of charge via theInternet at http://pubs.acs.org.

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Figure 3. Results of MDA-MB-231 exposure to the MARS. Panel (a)shows fluorescent microscope images (1, 3, and 5) and fluorescent imageswith differential interference contrast (2, 4, and 6). Color scheme: green,fluorescently labeled MARS; red, doxorubicin (DOX); yellow, merged greenand red. MARS nanoparticles containing DOX were taken up into the cells,but before the AC field was applied, no drug release (images 1 and 2) andnegligible cell death [∼5%; panel (b), left bar] occurred. Images 3 and 4show the effects of the magnetic field on MARS nanoparticles without DOXin the pores. Heating from the particles accounted for 16% of the cell killing[panel (b), middle bar]. Images 5 and 6 demonstrate DOX release after a 5min AC field exposure, which caused 37% of the cell death [panel b, rightbar]. The arrows in image 6 indicate the location of apoptotic cells.

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